Type 3 secretion is a common virulence mechanism utilized by many Gram-negative bacteria. These pathogens require these specialized secretion systems for survival in the host organism, therefore type 3 secretion systems are a good target for antimicrobial agents and also represent promising vaccine candidates. In type 3 secretion, bacteria bind to host cells and through a needle-like appendage translocate cytotoxic effector proteins directly into the cytosol of host cells. However upon host cell contact, the mechanisms by which secretion is triggered, effectors are efficiently translocated into the cell, as well as the intracellular trafficking of effectors within the cell are completely unknown. It is hypothesized that host cell factors play a critical role in all of these aspects contributing to host cell intoxication associated with type 3 secretion. To investigate this, the following proposed studies will ask whether RNAi knockdown of host cell factors inhibits a specific activity of a type 3 delivered effector from Yersinia pseudotuberculosis. The Yersinia effector YopE is a Rho GTPase activating protein, which stimulates the hydrolysis of GTP bound to the Rho family proteins. RNAi knockdown of host cell factors that aid in type 3 delivered YopE intoxication should therefore block the GTPase activation activity of YopE. A pooled unbiased whole genome RNAi screen will be performed using fluorescence resonance energy transfer to measure the activation state of a Rho GTPase biosensor in cells after infection with a Yersinia strain that secretes YopE. Fluorescence activated cell sorting will be used to positively select RNAi depleted cells that block YopE cytotoxicity in order to subsequently identify the targeted genes by microarray analysis. Additional experiments will characterize the mechanisms the identified host cell genes block type 3 mediated cytotoxicity. Studies will be performed to analyze whether these candidate genes specifically inhibit the activity of YopE or target common mechanisms required by all effectors suggesting a contribution to the process of type 3 secretion. PUBLIC HEALTH RELEVANCE: The innovative studies outlined in this application will provide a breadth of information about the role of the host cell in type 3 secretion mediated cytotoxicity by Yersinia as well as many other gram-negative bacteria that utilize these secretion systems as a virulence mechanism. Elucidation of host factors and pathways important to the function of type 3 secretion systems could provide new alternative targets for drug discovery to use as treatment options for these pathogenic bacteria.